Project Summary Autoreactive B cells play critical roles in many autoimmune diseases. Multiple immune tolerance checkpoints exist to remove autoreactive B cells or keep them under control. Defects in these checkpoints constitute the basis for the development of autoimmune diseases. Despite intensive study, our understanding of these checkpoints remains incomplete and fragmentary. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate a large diversity of biological processes. Hundreds of miRNAs are expressed in the immune system. While some miRNAs have been shown to play important roles in lymphocyte development and function, the roles of miRNAs in controlling immune tolerance remains poorly understood. We performed in vivo functional analysis of hundreds of miRNAs in the recently established IgMb-macroself mouse model and identified miR-148a as an important regulator of B cell tolerance and autoimmunity (Nature Immunology 17:433-40, 2016). Further molecular analysis identified 119 target genes regulated by miR-148a in immature B cells. We examined 4 of these target genes and demonstrated that 3 of them (Gadd45a, Bim and Pten) regulate B cell tolerance. The remaining 115 target genes have not been previously implicated in regulating B cell tolerance or autoimmunity. As miRNAs are thought to exert their functions by suppressing the expression of multiple target genes, we hypothesize that among the remaining 115 target genes, there are additional B cell tolerance regulators yet to be identified. In this proposal, we will perform functional analysis of these 115 target genes to identify novel regulators of B cell tolerance (Aim 1). In our pilot experiments, we identified B4galt5 as a strong positive hit, whose knockdown protects WEHI-231 cells from BCR engagement-induced apoptosis to the same degree as knockdown of Bim, a well established B cell tolerance regulator. B4galt5 is a critical enzyme that generates lactosylceramide (LacCer), a key intermediate in the glycolipid biosynthesis pathway. Glycolipids are major components of glycolipid enriched microdomains (GEM) of the outer leaflet of plasma membrane, which are also known as lipid rafts. We hypothesize that by controlling the glycolipid composition of lipid rafts, B4galt5 controls BCR signaling and B cell tolerance. Indeed, mutant mice deficient of B3gnt5, the enzyme that synthesizes lacto and neolacto series of glycolipids from LacCer, exhibited autoimmune symptoms such as splenomegaly and enlarged lymph nodes, as well as elevated BCR signaling and antibody responses. In Aim 2, we will investigate the roles of the glycolipid biosynthesis pathway in regulating B cell tolerance and autoimmunity, focusing on studying the functions and mechanisms of B4galt5 and B3gnt5 in controlling these processes.